EP0078734A1 - Procedure for immunological determinations using two antibodies, the second antibody being purified by affinity chromatography, and supports therefor - Google Patents

Procedure for immunological determinations using two antibodies, the second antibody being purified by affinity chromatography, and supports therefor Download PDF

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Publication number
EP0078734A1
EP0078734A1 EP82401955A EP82401955A EP0078734A1 EP 0078734 A1 EP0078734 A1 EP 0078734A1 EP 82401955 A EP82401955 A EP 82401955A EP 82401955 A EP82401955 A EP 82401955A EP 0078734 A1 EP0078734 A1 EP 0078734A1
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Prior art keywords
antibody
tubes
gamma
purified
affinity chromatography
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EP0078734B1 (en
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Gérard Trouyez
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Bme Te Charbonnieres-Les-Bains Frankrijk Firma
Biomerieux SA
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Biomerieux SA
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    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L9/00—Supporting devices; Holding devices
    • B01L9/06—Test-tube stands; Test-tube holders
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
    • G01N33/544—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals the carrier being organic
    • G01N33/545—Synthetic resin
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/74—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving hormones or other non-cytokine intercellular protein regulatory factors such as growth factors, including receptors to hormones and growth factors

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  • the subject of the invention is new supports intended for immunological assays of an antigen or a hapten, the coating of which fixed, by adsorption or covalent bonding, on the support which most often has the form of a tube in plastic, is a second antibody (that is to say an antibody directed against the first antibody) highly purified by affinity chromatography; the invention also relates to a process for the preparation of these supports and in particular of tubes.
  • These which can be supplied numbered and placed on racks so as to be ready for use, are stable for at least 15 days at 37 ° C and 6 months at + 4 ° C in their packaging.
  • a second antibody is used which is directed against the first antibody and therefore reacts with it, to separate the free antigen from that bound to the first antibody.
  • the second antibody is used in the free state (Morgan and Lazarow 1962, Utiger and Col. 1962).
  • a precipitation accelerator polyethylene glycol, ethanol, ammonium sulfate etc
  • a precipitation accelerator polyethylene glycol, ethanol, ammonium sulfate etc
  • the second antibody (Ab 2 ) is fixed on the solid phase, and this is incubated with a solution of the first antibody (Ab) and the solution containing the antigen to be assayed (Ag) to which we add -then the labeled antigen (Ag *).
  • Ab-Ag and Ab-Ag * complexes are formed which bind to Ab 2 on the solid phase, the non-combined (labeled and unlabeled) antigen remaining in the liquid phase.
  • the second antibody is usually obtained by repeated injections of plasma or blood serum from an animal or man into an animal of another species: the serum of the latter contains an antibody (gamma globulin) directed against gamma globulins of the first animal or man.
  • the gamma globulins (Ab 2 ) of the serum of the immunized animal must be separated and purified before being fixed on the solid phase.
  • the gamma globulins of the second antibody are purified only by precipitation with sodium sulfate followed by molecular filtration on Sephadex G-200 gel before being adsorbed on polystyrene beads.
  • the handling of the balls lacks practicability compared to that of the tubes. In addition, they represent an additional reagent to handle.
  • the gamma globulins of the second antibody are purified only by precipitation with ammonium sulphate followed by chromatography on DEAE cellulose before being adsorbed on polystyrene tubes. These tubes were used only for the determination of the somatotropic chorionic hormone (HCS) according to a procedure characterized by incubations at 37 ° C.
  • HCS somatotropic chorionic hormone
  • a gel preferably a Sepharose 4B or Ultragel ACA 22 gel
  • rabbit gamma globulins have been fixed.
  • the actual affinity chromatography is then carried out as follows:
  • the sheep serum (100 ml for example) rabbit anti-gamma globulin is introduced into the column and eluted with a sodium phosphate buffer at pH 7 , 4 until the return of the baseline of the chromatogram.
  • the rabbit anti-gamma globulin gamma globulins are then eluted by a buffer providing a sudden change in pH and conductivity of the column (for example, a glycine-sodium hydroxide buffer of pH 2.8).
  • Second step Example of fixation by adsorption on polyethylene tubes of sheep gamma globulin anti-rabbit gamma globulin globulins purified by affinity chromatography:
  • the eluate obtained above is incubated (30 to 100 ⁇ l / ml) in each of the tubes; these are then rinsed successively with a sodium chloride solution at 9 ° / oo and with a protein solution.
  • the tubes thus treated are dried by lyophilization or any other process before being packaged in a sachet in the presence of a desiccant.
  • the supports coated with a second antibody, according to the invention, are used for immunological and more particularly radio-immunological assays of hormones such as human follicle stimulating hormone (hFSH) and human luteinizing hormone (hLH ).
  • hormones such as human follicle stimulating hormone (hFSH) and human luteinizing hormone (hLH ).
  • tubes can be grouped together in reagent systems or "kits" (for immunoassays), on racks made of plastic materials, preferably thermoformed.
  • FIG. 1 An embodiment of a tube rack according to the invention is shown in Figures 1, 2 and 3 below.
  • Figure 2 shows a top view, Figure 3 a transverse view and Figure 1 a longitudinal section along the axis I-I.
  • the rack consists of a base 1 having contiguous, circular cells, having a thin channel from top to bottom intended to facilitate the introduction of the tubes, and of a cover 2 also made of plastic materials, preferably thermoformed, snap into the base, thanks to the snap rings 3.
  • tubes ready for use that is to say dry, numbered and arranged in racks, which considerably improves the implementation of immunoassays.

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Abstract

1. Process for immuno assay of antigens, employing two antibodies, consisting in fixing a second antibody on a solid phase, in incubating the latter with a solution of thu first antibody and the solution containing the antigen to be assayed to which the marked antigen is then added, characterized in that a second antibody, rendered insoluble and which the previously been purified by affinity chromatography, is used.

Description

L'invention a pour objet de nouveaux supports destinés à des dosages immunologiques d'un antigène ou d'un haptène, dont le revêtement fixé, par adsorption ou liaison covalente, sur le support qui a le plus souvent la forme d'un tube en matière plastique, est un deuxième anticorps (c'est-à-dire un anticorps dirigé contre le premier anticorps) hautement purifié par chromatographie d'affinité ; l'invention a également pour objet un procédé de préparation de ces supports et en particulier de tubes. Ceux-ci, qui peuvent être fournis numérotés et disposés sur des portoirs de manière à être prêts à l'emploi, sont stables au moins 15 jours à 37°C et 6 mois à +4°C dans leur emballage.The subject of the invention is new supports intended for immunological assays of an antigen or a hapten, the coating of which fixed, by adsorption or covalent bonding, on the support which most often has the form of a tube in plastic, is a second antibody (that is to say an antibody directed against the first antibody) highly purified by affinity chromatography; the invention also relates to a process for the preparation of these supports and in particular of tubes. These, which can be supplied numbered and placed on racks so as to be ready for use, are stable for at least 15 days at 37 ° C and 6 months at + 4 ° C in their packaging.

Ils permettent de simplifier la réalisation des dosages immunologiques d'antigènes et d'haptènes.They simplify the carrying out of immunological assays of antigens and haptens.

Dans de nombreuses méthodes de dosage immunologique et plus particulièrement radio-immunologique, on utilise un deuxième anticorps dirigé contre le premier anticorps et donc réagissant avec lui, pour séparer l'antigène libre de celui lié au premier anticorps.In many immunological and more particularly radio-immunological assay methods, a second antibody is used which is directed against the first antibody and therefore reacts with it, to separate the free antigen from that bound to the first antibody.

Dans les méthodes immunoprécipitantes, le deuxième anticorps est utilisé à l'état libre (Morgan et Lazarow 1962, Utiger et Col. 1962).In immunoprecipitating methods, the second antibody is used in the free state (Morgan and Lazarow 1962, Utiger and Col. 1962).

Dans les méthodes d'immunoprecipitation accélérée, un accélérateur de précipitation (polyéthylèneglycol, éthanol, sulfate d'ammonium etc) est ajouté au deuxième anticorps, de manière à réduire la durée de l'immunoprécipitation à 15 ou 30 minutes (Martin et Landon 1974).In accelerated immunoprecipitation methods, a precipitation accelerator (polyethylene glycol, ethanol, ammonium sulfate etc) is added to the second antibody, so as to reduce the duration of immunoprecipitation to 15 or 30 minutes (Martin and Landon 1974) .

Dans les méthodes utilisant un immunoadsorbant microparticulaire, le deuxième anticorps est soit :

  • - préincubé à un premier anticorps ;
  • - couplé à de la cellulose microcristalline ou à du Sépharose 4B (DASP) selon WIDE, à du gel d'acrylamide, à des particules de latex etc;
  • - couplé à des polymères associés à de l'oxyde de fer (Nye et col. 1976).
In methods using a microparticulate immunoadsorbent, the second antibody is either:
  • - preincubated with a first antibody;
  • - coupled to microcrystalline cellulose or Sepharose 4B (DASP) according to WIDE, to acrylamide gel, to latex particles etc;
  • - coupled with polymers associated with iron oxide (Nye et al. 1976).

A ce type de méthode doivent être assimilées les techniques faisant appel à la protéine A du staphylocoque doré qui joue un rôle comparable au deuxième anticorps (Jonsson et Kronvall 1973).To this type of method should be assimilated the techniques using protein A of staphylococcus aureus which plays a role comparable to the second antibody (Jonsson and Kronvall 1973).

Toutes ces méthodes présentent deux inconvénients essentiels :

  • - le pipetage d'un réactif en suspension constitue souvent une source d'hétérogénéité dans les dosages. Seules les méthodes immunoprécipitantes simples ne présentent pas ce premier inconvénient ;
  • - la nécessité de faire appel à une étape de centrifugation ou de microfiltration pour séparer la fraction d'antigène libre de celle liée. Seule la méthode utilisant des particules d'oxyde de fer évite cet inconvénient en réalisant la séparation dans un champ magnétique ; cela implique par contre la nécessité de se procurer des équipements supplémentaires.
All these methods have two essential drawbacks:
  • - the pipetting of a suspension reagent often constitutes a source of heterogeneity in the assays. Only simple immunoprecipitating methods do not have this first drawback;
  • - The need to use a centrifugation or microfiltration step to separate the free antigen fraction from that bound. Only the method using iron oxide particles avoids this drawback by carrying out the separation in a magnetic field; on the other hand, this implies the need to obtain additional equipment.

La transposition des méthodes en phase solide selon Catt et Tregear (1967) au couplage du deuxième anticorps supprime ce deuxième inconvénient.The transposition of the solid phase methods according to Catt and Tregear (1967) to the coupling of the second antibody eliminates this second drawback.

Dans la méthode RIA, le deuxième anticorps (Ab2) est fixé sur la phase solide, et celle-ci est incubée avec une solution du premier anticorps (Ab) et la solution contenant l'antigène à doser (Ag) à laquelle on ajoute-ensuite l'antigène marqué (Ag*). Il se forme des complexes Ab-Ag et Ab-Ag* qui se fixent au Ab2 sur la phase solide, l'antigène (marqué et non-marqué) non-combiné restant dans la phase liquide.In the RIA method, the second antibody (Ab 2 ) is fixed on the solid phase, and this is incubated with a solution of the first antibody (Ab) and the solution containing the antigen to be assayed (Ag) to which we add -then the labeled antigen (Ag *). Ab-Ag and Ab-Ag * complexes are formed which bind to Ab 2 on the solid phase, the non-combined (labeled and unlabeled) antigen remaining in the liquid phase.

Le deuxième anticorps est habituellement obtenu par injections répétées du plasma ou du sérum sanguin d'un animal ou de l'homme à un animal d'une autre espèce : le sérum de celui-ci contient un anticorps (gamma-globulines) dirigé contre les gamma- globulines du premier animal ou de l'homme. Les gamma-globulines (Ab2) du sérum de l'animal immunisé doivent être séparées et purifiées avant d'être fixées sur la phase solide.The second antibody is usually obtained by repeated injections of plasma or blood serum from an animal or man into an animal of another species: the serum of the latter contains an antibody (gamma globulin) directed against gamma globulins of the first animal or man. The gamma globulins (Ab 2 ) of the serum of the immunized animal must be separated and purified before being fixed on the solid phase.

Dans la méthode de ZIOLA B.R. et col. (1977), les gamma- globulines du deuxième anticorps (sérum de mouton, anti-gamma-globuline de lapin) sont purifiées seulement par une précipitation au sulfate de sodium suivie d'une filtration moléculaire sur gel Sephadex G-200 avant d'être adsorbées sur des billes de polystyrène. Cependant, la manipulation des billes manque de praticabilité par rapport à celle des tubes. De plus, elles représentent un réactif supplémentaire à manipuler.In the method of ZIOLA BR et al. (1977), the gamma globulins of the second antibody (sheep serum, rabbit anti-gamma globulin) are purified only by precipitation with sodium sulfate followed by molecular filtration on Sephadex G-200 gel before being adsorbed on polystyrene beads. However, the handling of the balls lacks practicability compared to that of the tubes. In addition, they represent an additional reagent to handle.

Dans la méthode de Cocola F. et col. (1973), les gamma- globulines du deuxième anticorps (sérum de chèvre, anti-gamma-globuline de lapin) sont purifiées seulement par une précipitation au sulfate d'ammonium suivie d'une chromatographie sur DEAE cellulose avant d'être adsorbées sur des tubes en polystyrène. Ces tubes ont servi uniquement pour le dosage de l'hormone chorionique somatotrope (HCS) selon un mode opératoire caractérisé par des incubations à 37°C et une addition séquentielle des réactifs (pipetage successif du sérum anti-HCS et des échantillons à doser, suivie de 4 heures d'incubation à 37°C puis d'une addition de HCS marqué à l'iode 125, suivie d'une incubation de 14 heures à 37°C). La stabilité de ces tubes ne dépasse pas 2 mois à +4°C. De plus, ce travail n'a pas permis le développement de réactifs commerciaux.In the method of Cocola F. et al. (1973), the gamma globulins of the second antibody (goat serum, rabbit anti-gamma globulin) are purified only by precipitation with ammonium sulphate followed by chromatography on DEAE cellulose before being adsorbed on polystyrene tubes. These tubes were used only for the determination of the somatotropic chorionic hormone (HCS) according to a procedure characterized by incubations at 37 ° C. and a sequential addition of the reagents (successive pipetting of the anti-HCS serum and of the samples to be assayed, followed 4 hours incubation at 37 ° C followed by addition of HCS labeled with iodine 125, followed by a 14 hour incubation at 37 ° C). The stability of these tubes does not exceed 2 months at + 4 ° C. In addition, this work did not allow the development of commercial reagents.

Dans la méthode selon l'invention, les gamma-globulines du deuxième anticorps sont purifiées par chromatographie d'affinité avant d'être fixées sur les supports, en particulier les tubes qui sont de préférence en polyéthylène. Il en résulte l'obtention d'immunoadsorbants beaucoup plus performants se prêtant lors des dosages :

  • - à l'addition en une seule étape, sans incubation intermédiaire, de tous les réactifs successivement, le premier anticorps étant ajouté en dernier en tant que réactif déclanchant ;
  • - à une incubation durant de quelques heures (méthode en cinétique) à 24 heures (méthode à l'équilibre) à la température du laboratoire, ou autre température.
In the method according to the invention, the gamma globulins of the second antibody are purified by affinity chromatography before being fixed on the supports, in particular the tubes which are preferably made of polyethylene. This results in the obtaining of much more efficient immunoadsorbents suitable for assays:
  • - the addition in a single step, without intermediate incubation, of all the reagents successively, the first antibody being added last as a triggering reagent;
  • - to an incubation lasting from a few hours (kinetic method) to 24 hours (equilibrium method) at laboratory temperature, or other temperature.

Le procédé de préparation des tubes revêtus d'un deuxième anticorps, selon l'invention, comprend donc deux étapes :

  • 1) Obtention des gamma-globulines du deuxième anticorps hautement purifiées par chromatographie d'affinité sur une colonne constituée par un gel sur lequel ont été fixées les gamma-globulines contre lesquelles est dirigé l'anticorps à purifier ;
  • 2) fixation de ces gamma-globulines sur les tubes en polymères (polyéthylène, polystyrène et autres).
The process for preparing the tubes coated with a second antibody, according to the invention, therefore comprises two stages:
  • 1) Obtaining gamma globulins of the second highly purified antibody by affinity chromatography on a column constituted by a gel on which the gamma globulins against which the antibody to be purified is directed are fixed;
  • 2) fixation of these gamma globulins on polymer tubes (polyethylene, polystyrene and others).

Première étape : Exemple de purification de gamma-globulines de sérum de mouton anti-gamma-globulines de lapin :First step: Example of purification of gamma-globulin from sheep serum anti-gamma-globulin from rabbit:

On utilise, pour la chromatographie d'affinité, un gel (de préférence un gel de Sepharose 4B ou d'Ultragel ACA 22) sur lequel ont été fixés des gamma-globulines de lapin.Use is made, for affinity chromatography, of a gel (preferably a Sepharose 4B or Ultragel ACA 22 gel) to which rabbit gamma globulins have been fixed.

La chromatographie d'affinité proprement dite s'effectue alors de la façon suivante : On introduit le sérum de mouton (100 ml par exemple) anti-gamma-globulines de lapin dans la colonne et on élue par un tampon phosphate de sodium à pH 7,4 jusqu'au retour de la ligne de base du chromatogramme. Les gamma-globulines de mouton anti-gammaglobulines de lapin sont ensuite éluées par un tampon apportant une variation brutale de pH et de conductivité de la colonne (par exemple, un tampon glycine-soude de pH 2,8).The actual affinity chromatography is then carried out as follows: The sheep serum (100 ml for example) rabbit anti-gamma globulin is introduced into the column and eluted with a sodium phosphate buffer at pH 7 , 4 until the return of the baseline of the chromatogram. The rabbit anti-gamma globulin gamma globulins are then eluted by a buffer providing a sudden change in pH and conductivity of the column (for example, a glycine-sodium hydroxide buffer of pH 2.8).

Après équilibrage de l'éluat à pH 7,4, on le fixe sur les tubes.After balancing the eluate at pH 7.4, it is fixed on the tubes.

Deuxième étape : Exemple de fixation par adsorption sur des tubes en polyéthylène de gamma-globulines de mouton anti-gammablobulines de lapin purifiées par chromatographie d'affinité :Second step: Example of fixation by adsorption on polyethylene tubes of sheep gamma globulin anti-rabbit gamma globulin globulins purified by affinity chromatography:

On fait incuber l'éluat obtenu précédemment (30 à 100 µl/ml) dans chacun des tubes ; ceux-ci sont ensuite rincés successivement par une solution de chlorure de sodium à 9°/oo et par une solution protéique. Les tubes ainsi traités sont séchés par lyophilisation ou tout autre procédé avant d'être conditionnés en sachet en présence d'un déshydratant.The eluate obtained above is incubated (30 to 100 μl / ml) in each of the tubes; these are then rinsed successively with a sodium chloride solution at 9 ° / oo and with a protein solution. The tubes thus treated are dried by lyophilization or any other process before being packaged in a sachet in the presence of a desiccant.

L'originalité de ces tubes tient :

  • - à leur stabilité dans leur conditionnement au moins 15 jours à 37°C et 6 mois à +4°C ;
  • - à leur performance de reproductibilité dans les dosages.
The originality of these tubes is:
  • - their stability in their packaging at least 15 days at 37 ° C and 6 months at + 4 ° C;
  • - their reproducibility performance in the assays.

Les supports revêtus d'un deuxième anticorps, selon l'invention, sont utilisés pour les dosages immunologiques et plus particulièrement radio-immunologiques, d'hormones telles que l'hormone folliculo- stimulante humaine (hFSH) et l'hormone lutéinisante humaine (hLH). Premier exemple d'application des tubes selon l'invention au dosageThe supports coated with a second antibody, according to the invention, are used for immunological and more particularly radio-immunological assays of hormones such as human follicle stimulating hormone (hFSH) and human luteinizing hormone (hLH ). First example of application of the tubes according to the invention to the assay

radio-immunologique de la hFSH :hFSH radioimmunoassay:

Le système de réactifs contient

  • 2 flacons de réactif R1 , 125I - hFSH (≤ 0,75 µCi)
  • 7 flacons de réactif R2 : hFSH étalon à des concentrations comprises entre 0 et 40 ng/ml
  • 2 flacons de réactif R3 : sérum de lapin anti-hFSH
  • 2 portoirs de 48 tubes selon l'invention de réactif R4 : (gamma- globulines de mouton anti-gamma-globulines de lapin)
  • 1 flacon de réactif R5 : diluant (sans hFSH)
  • 1 flacon de réactif R6 : tampon PBS-BSA ph 7,4 → 50 ml
  • 1 flacon de réactif R7
  • 1 flacon de réactif R8 sérums de contrôle humain acon réactif
The reagent system contains
  • 2 bottles of reagent R1, 125 I - hFSH (≤ 0.75 µCi)
  • 7 bottles of reagent R2: standard hFSH at concentrations between 0 and 40 ng / ml
  • 2 bottles of reagent R3: anti-hFSH rabbit serum
  • 2 racks of 48 tubes according to the invention of reagent R4: (sheep gamma globulin anti-rabbit gamma globulin)
  • 1 bottle of reagent R5: diluent (without hFSH)
  • 1 bottle of reagent R6: PBS-BSA buffer ph 7.4 → 50 ml
  • 1 bottle of R7 reagent
  • 1 bottle of reagent R8 reagent human control sera

Deuxième exemple d'application des tubes selon l'invention au dosage radio-immunologique de la hLH :Second example of application of the tubes according to the invention to the radioimmunoassay of hLH:

Le système de réactifs contient :

  • 2 flacons de réactif R1 : 125I-hLH (≤ 0,75 µCi)
  • 7 flacons de réactif R2 : hLH étalon à des concentrations comprises entre 0 et 40 ng/ml
  • 2 flacons de réactif R3 : sérum de lapin anti-hLH
  • 2 portoirs de 48 tubes selon l'invention de réactif R4 : (gamma- globulines de mouton anti-gamma-globulines de lapin)
  • 1 flacon de réactif R5 : diluant (sans hLH)
  • 1 flacon de réactif R6 : tampon PBS-BSA pH 7,4 →50 ml
  • 1 flacon de réactif R7
  • 1 flacon de réactif R8 sérums de contrôle humain
The reagent system contains:
  • 2 vials of reagent R1: 125 I-hLH (≤ 0, 75 .mu.Ci)
  • 7 bottles of reagent R2: standard hLH at concentrations between 0 and 40 ng / ml
  • 2 bottles of reagent R3: anti-hLH rabbit serum
  • 2 racks of 48 tubes according to the invention of reagent R4: (sheep gamma globulin anti-rabbit gamma globulin)
  • 1 bottle of reagent R5: diluent (without hLH)
  • 1 bottle of reagent R6: PBS-BSA buffer pH 7.4 → 50 ml
  • 1 bottle of R7 reagent
  • 1 bottle of reagent R8 human control sera

L'originalité de ces deux systèmes de réactifs tient :

  • - à la simplicité de leur mode opératoire :
    • . tous les réactifs sont ajoutés successivement en une seule étape, sans incubation intermédiaire, le premier anticorps (anti-hLH ou anti-hFSH) étant ajouté en dernier ;
    • . l'incubation est de quelques heures à 24 heures à la température du laboratoire. Il n'y a pas d'étape de centrifugation.
  • - à la conception du dosage :
    • le fait d'ajouter le premier anticorps en dernier, contrairement au protocole de Cocola permet à ce réactif de jouer un rôle de réactif déclenchant : ainsi, les réactions immunologiques du dosage démarrent au même moment dans tous les tubes. Le dosage peut ainsi être fait en cinétique ou à l'équilibre. Cet avantage existe de la même manière vis-à-vis des méthodes en tubes revêtus pour lesquelles un premier anticorps et non un deuxième anticorps est fixé au fond des tubes ;
    • . l'absence d'adsorption des traceurs 125 I-hLH ou 125I-hFSH dans les tubes coat RIA dans les conditions du dosage ainsi que l'aspiration du milieu réactionnel en fin d'incubation suivie d'un rinçage permet d'obtenir des "non-spécifiques" inférieurs à 1 %.
The originality of these two reagent systems is:
  • - the simplicity of their operating mode:
    • . all the reagents are added successively in a single step, without intermediate incubation, the first antibody (anti-hLH or anti-hFSH) being added last;
    • . incubation is from a few hours to 24 hours at laboratory temperature. There is no centrifugation step.
  • - the design of the dosage:
    • adding the first antibody last, unlike the Cocola protocol, allows this reagent to act as a triggering reagent: thus, the immunological reactions of the assay start at the same time in all the tubes. The assay can thus be done in kinetics or in equilibrium. This advantage exists in the same way with respect to coated tube methods for which a first antibody and not a second antibody is attached to the bottom of the tubes;
    • . the absence of adsorption of the 125 I-hLH or 125 I-hFSH tracers in the RIA coat tubes under the assay conditions as well as the aspiration of the reaction medium at the end of incubation followed by rinsing makes it possible to obtain "non-specific" less than 1%.

L'emploi des supports selon l'invention peut être étendu au dosage radio-immunologique, enzymo-immunologique, colorimétrique ou fluorescent de produits tels que :

  • - les haptènes : stéroïdes, vitamines, médicaments, etc.
  • - les macromolécules : protéines telles que des hormones (prolactine, hormone thyréostimulante ...) des protéines sériques (protéines liantes, anticorps ...) des protéines tissulaires dans les matériaux biologiques tels que le sérum, le plasma, l'urine, des virus végétaux, etc.
The use of the supports according to the invention can be extended to radioimmunological, enzymo-immunological, colorimetric or fluorescent assay of products such as:
  • - haptens: steroids, vitamins, drugs, etc.
  • - macromolecules: proteins such as hormones (prolactin, thyroid-stimulating hormone ...) serum proteins (binding proteins, antibodies ...) tissue proteins in biological materials such as serum, plasma, urine, plant viruses, etc.

Pour faciliter l'emploi des tubes selon l'invention, ils peuvent être regroupés dans les systèmes de réactifs ou "kits" (pour les dosages immunologiques), sur des portoirs réalisés en matériaux plastiques, de préférence thermoformés.To facilitate the use of the tubes according to the invention, they can be grouped together in reagent systems or "kits" (for immunoassays), on racks made of plastic materials, preferably thermoformed.

Une forme de réalisation d'un portoir de tubes selon l'invention est représentée dans les figures 1, 2 et 3 ci-après.An embodiment of a tube rack according to the invention is shown in Figures 1, 2 and 3 below.

La figure 2 représente une vue de dessus, la figure 3 une vue transversale et la figure 1 une coupe longitudinale selon l'axe I-I.Figure 2 shows a top view, Figure 3 a transverse view and Figure 1 a longitudinal section along the axis I-I.

Le portoir est constitué d'une base 1 présentant des alvéoles contiguës, circulaires, ayant un fin canal de haut en bas destiné à faciliter l'introduction des tubes, et d'un couvercle 2 également réalisé en matériaux plastiques, de préférence thermoformés, venant s'enclipser dans la base, grâce aux joncs d'enclipsage 3.The rack consists of a base 1 having contiguous, circular cells, having a thin channel from top to bottom intended to facilitate the introduction of the tubes, and of a cover 2 also made of plastic materials, preferably thermoformed, snap into the base, thanks to the snap rings 3.

On constitue ainsi un ensemble compact ayant les avantages suivants :

  • a) les tubes étant jointifs sont parfaitement alignés dans le pfirtoir, facilitant ainsi les opérations de pipetage et de lavage.
  • b) La bonne tenue de ces tubes permet leur manipulation aisée et notamment retournement pour toute opération de vidange et égouttage.
  • c) Le couvercle disposable peut être maintenu en position haute pour permettre le séchage par lyophilisation ou tout autre moyen. Le clipsage peut alors se faire dans la machine même par rapprochement des plateaux supports. De plus, un gaz neutre peut être introduit dans l'emballage afin de protéger le produit jusqu'à sa mise en conditionnement étanche définitif.
  • d) La forme de coque du couvercle offre la possibilité d'operculer au moyen d'un film barrière thermosoudable. Un blister étanche est ainsi réalisé.
  • e) Les tubes, au nombre de 48, sont numérotés et prêts à l'emploi. Ce sont des tubes à hémolyse en polyéthylène, polystyrène ou toute autre matière plastique.
  • f) Le modèle de portoir est applicable à toute forme de tube, ou contenant, destiné à des répartitions et lavages en série.
This constitutes a compact assembly having the following advantages:
  • a) the tubes being joined are perfectly aligned in the pfirtoir, thus facilitating the pipetting and washing operations.
  • b) The good behavior of these tubes allows their easy handling and in particular turning over for any emptying and draining operation.
  • c) The disposable cover can be kept in the high position to allow drying by lyophilization or any other means. Clipping can then be done in the machine even by bringing the support plates together. In addition, a neutral gas can be introduced into the packaging in order to protect the product until it is finally sealed.
  • d) The shell shape of the cover offers the possibility of sealing with a heat-sealable barrier film. A waterproof blister is thus produced.
  • e) The tubes, 48 in number, are numbered and ready to use. These are hemolysis tubes made of polyethylene, polystyrene or any other plastic material.
  • f) The rack model is applicable to any form of tube, or container, intended for distribution and washing in series.

En résumé, on obtient ainsi selon l'invention des tubes prêts à l'emploi, c'est-à-dire secs, numérotés et disposés en portoirs, ce qui améliore de manière considérable la mise en oeuvre des dosages immunologiques.In summary, thus obtained according to the invention tubes ready for use, that is to say dry, numbered and arranged in racks, which considerably improves the implementation of immunoassays.

Claims (12)

1. Supports en matière plastique revêtus d'un deuxième anticorps, pour les dosages immunologiques, caractérisés en ce que le revêtement est constitué par un deuxième anticorps hautement purifié par chromatographie d'affinité, fixé par adsorption sur un polymère, de préférence un polyéthylène.1. Plastic supports coated with a second antibody, for immunoassays, characterized in that the coating consists of a second antibody highly purified by affinity chromatography, fixed by adsorption on a polymer, preferably a polyethylene. 2. Supports selon la revendication 1, caractérisé en ce que le deuxième anticorps est une gamma-globuline du sérum d'une espèce animale, anti-gamma-globuline du sérum d'une autre espèce animale ou de l'homme, purifiée par chromatographie sur des gamma-globulines de la deuxième espèce animale ou de l'homme.2. Supports according to claim 1, characterized in that the second antibody is a gamma globulin of the serum of an animal species, anti-gamma globulin of the serum of another animal species or of man, purified by chromatography on gamma globulins of the second animal or human species. 3. Supports selon l'une des revendications 1 et 2, caractérisés en ce que le deuxième anticorps est une gamma-globuline de mouton anti-gamma-globuline de lapin.3. Supports according to one of claims 1 and 2, characterized in that the second antibody is a gamma-globulin of sheep anti-gamma-globulin of rabbit. 4. Supports selon l'une des revendications 1 à 3, caractérisés en ce qu'ils ont la forme de tubes.4. Supports according to one of claims 1 to 3, characterized in that they have the form of tubes. 5. Procédé de préparation de supports selon l'une des revendications 1 à 4, caractérisé en ce qu'on traite le sérum immun contenant le deuxième anticorps par chromatographie d'affinité sur des gamma- globulines contre lesquelles est dirigé l'anticorps à purifier et que le deuxième anticorps purifié ainsi obtenu est fixé par adsorption sur le support en une matière plastique telle que polyéthylène ou polystyrène.5. Method for preparing supports according to one of claims 1 to 4, characterized in that the immune serum containing the second antibody is treated by affinity chromatography on gamma globulins against which the antibody to be purified is directed and that the second purified antibody thus obtained is fixed by adsorption on the support in a plastic material such as polyethylene or polystyrene. 6. Procédé selon la revendication 5, caractérisé en ce que, pour la chromatographie d'affinité, on utilise une colonne remplie d'un gel sur lequel sont fixées des gamma-globulines contre lesquelles est dirigé l'anticorps à purifier, et on fait passer sur cette colonne le sérum immun contenant l'anticorps à purifier, puis on élue par un tampon phosphate à pH 7,4 jusqu'au retour de la ligne de base du chromatogramme ; on élue ensuite les gamma-globulines du deuxième anticorps par un tampon apportant une variation brutale de pH et de conductivité de la colonne.6. Method according to claim 5, characterized in that, for the affinity chromatography, a column filled with a gel is used on which gamma globulins are fixed against which the antibody to be purified is directed, and one makes pass over this column the immune serum containing the antibody to be purified, then elute with a phosphate buffer at pH 7.4 until the return of the baseline of the chromatogram; the gamma globulins of the second antibody are then eluted with a buffer providing a sudden change in pH and conductivity of the column. 7. Procédé selon l'une des revendications 5 et 6, caractérisé en ce que l'éluat contenant les gamma-globulines du deuxième anticorps est équilibré à pH 7,4, dilué et incubé sur le support et en particulier dans les tubes où il se fixe par adsorption sur le polymère constituant la paroi du tube.7. Method according to one of claims 5 and 6, characterized in that the eluate containing the gamma globulins of the second antibody is balanced to pH 7.4, diluted and incubated on the support and in particular in the tubes where it is fixed by adsorption on the polymer constituting the wall of the tube. 8. Procédé de dosages immunologiques utilisant un deuxième anticorps en phase solide, caractérisé en ce qu'on utilise les tubes selon la revendication 4, et qu'on ajoute successivement les autres réactifs dans les tubes, en une seule étape, le premier anticoprs étant ajouté en dernier et servant de réactif déclenchant.8. A method of immunoassays using a second antibody in solid phase, characterized in that the tubes according to claim 4 are used, and that the other reagents are successively added to the tubes, in a single step, the first anticoprs being added last and used as a triggering reagent. 9. Procédé selon la revendication 8, caractérisé en ce qu'on dose l'hormone folléculo-stimulante et l'hormone lutéinisante humaines.9. Method according to claim 8, characterized in that the folleculo-stimulating hormone and the human luteinizing hormone are measured. 10. Procédé selon la revendication 9, caractérisé en ce qu'on réalise le dosage aussi bien à l'équilibre qu'en cinétique.10. Method according to claim 9, characterized in that the metering is carried out both at equilibrium and in kinetics. 11. Dispositif de présentation ou portoir pour les tubes selon la revendication 4, caractérisé en ce qu'il est constitué par une matière plastique thermoformée et comprend une base (1) présentant des alvéoles contiguës, circulaires, ayant un fin canal de haut en bas pour l'introduction des tubes et un couvercle (2) venant s'enclipser dans la base grâce à deux joncs d'enclipsage (3) qui permettent au couvercle d'être maintenu en position haute pour le séchage des tubes ou l'introduction d'un gaz de protection ou en position basse pour operculer les tubes et permettre leur retournement.11. Presentation device or rack for tubes according to claim 4, characterized in that it is constituted by a thermoformed plastic material and comprises a base (1) having contiguous, circular cells, having a fine channel from top to bottom for the insertion of the tubes and a cover (2) which snaps into the base thanks to two snap rings (3) which allow the cover to be held in the high position for drying the tubes or the introduction of '' a protective gas or in the low position to seal the tubes and allow them to turn over. 12. Dispositif selon la revendication 11, caractérisé en ce qu'il comprend 48 tubes numérotés et prêts à l'emploi.12. Device according to claim 11, characterized in that it comprises 48 numbered tubes and ready for use.
EP82401955A 1981-10-29 1982-10-25 Procedure for immunological determinations using two antibodies, the second antibody being purified by affinity chromatography, and supports therefor Expired EP0078734B1 (en)

Priority Applications (1)

Application Number Priority Date Filing Date Title
AT82401955T ATE22997T1 (en) 1981-10-29 1982-10-25 METHODS OF IMMUNOLOGICAL DETERMINATION USING TWO ANTIBODIES, THE SECOND ANTIBODY PURIFIED BY AFFINITY CHROMATOGRAPHY, AND CARRIER THEREFOR.

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
FR8120347 1981-10-29
FR8120347A FR2515827B1 (en) 1981-10-29 1981-10-29 NOVEL TUBES COATED WITH A SECOND HIGHLY PURIFIED ANTIBODY, READY TO USE, FOR IMMUNOLOGICAL ASSAYS, AND THEIR PREPARATION METHOD

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EP0078734A1 true EP0078734A1 (en) 1983-05-11
EP0078734B1 EP0078734B1 (en) 1986-10-15

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JP (1) JPS58144749A (en)
AT (1) ATE22997T1 (en)
DE (1) DE3273844D1 (en)
ES (1) ES516244A0 (en)
FR (1) FR2515827B1 (en)

Cited By (4)

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EP0155224A2 (en) * 1984-03-15 1985-09-18 CHROMAGENICS, Inc. Solid-borne complex bearing chromagen responsive functionality for antibody, antigen, receptor, or ligand detection
FR2579757A1 (en) * 1985-04-02 1986-10-03 Takara Shuzo Co METHOD FOR DETERMINING HEMOGLOBIN IN FECAL MATERIALS
EP0213653A1 (en) * 1985-08-19 1987-03-11 Akzo N.V. Device for the carrying out of an immunochemical determination
CH664094A5 (en) * 1984-08-30 1988-02-15 Treff Ag Clip-on lid covers arrayed containers - projecting from apertured carrier plate

Families Citing this family (2)

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JPS6082966A (en) * 1983-10-14 1985-05-11 Amano Pharmaceut Co Ltd Assay of antigen
US4608231A (en) * 1984-12-12 1986-08-26 Becton, Dickinson And Company Self-contained reagent package device for an assay

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US4048298A (en) * 1975-02-25 1977-09-13 Rohm And Haas Company Solid phase double-antibody radioimmunoassay procedure
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Cited By (5)

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EP0155224A2 (en) * 1984-03-15 1985-09-18 CHROMAGENICS, Inc. Solid-borne complex bearing chromagen responsive functionality for antibody, antigen, receptor, or ligand detection
EP0155224A3 (en) * 1984-03-15 1987-05-06 CHROMAGENICS, Inc. Solid-borne complex bearing chromagen responsive functionality for antibody, antigen, receptor, or ligand detection
CH664094A5 (en) * 1984-08-30 1988-02-15 Treff Ag Clip-on lid covers arrayed containers - projecting from apertured carrier plate
FR2579757A1 (en) * 1985-04-02 1986-10-03 Takara Shuzo Co METHOD FOR DETERMINING HEMOGLOBIN IN FECAL MATERIALS
EP0213653A1 (en) * 1985-08-19 1987-03-11 Akzo N.V. Device for the carrying out of an immunochemical determination

Also Published As

Publication number Publication date
ATE22997T1 (en) 1986-11-15
JPS58144749A (en) 1983-08-29
ES8401261A1 (en) 1983-11-16
FR2515827A1 (en) 1983-05-06
DE3273844D1 (en) 1986-11-20
ES516244A0 (en) 1983-11-16
FR2515827B1 (en) 1985-12-27
EP0078734B1 (en) 1986-10-15

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